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The purpose of this study was to structurally characterize the major lipid species present in human meibomian gland secretions (MGS) of individual subjects by means of ion trap atmospheric pressure ionization mass spectrometry analysis (API MSn). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MSn detection of the analytes (HPLC API MSn). The major precursor ions were isolated and subjected to further sequential fragmentation in MSn experiments, and their fragmentation patterns were compared with those of authentic lipid standards. Multiple precursor ions were observed in the positive-ion mode. Among those, previously identified cholesterol (Chl; m/z 369; [M − H2O + H]+) and oleic acid (OA; m/z 283; [M + H]+) were found. The other major compounds of the general molecular formula CnH2n-2O2 were consistent with wax esters (WEs), with OA as fatty acyl component. Accompanying them were two homologous series of compounds that fit the molecular formulas CnH2n-4O2 and CnH2nO2. Subset 2 was found to be a homolog series of linoleic acid-based WEs, whereas subset 3 was, apparently, a mixture of stearic acid-based WEs. HPLC API MSn analysis revealed the presence of large quantities of cholesteryl esters (Chl-Es) in all of the tested samples. Less than 0.1% (w/w) of oleamide was detected in human MGS. In the negative-ion mode, three major compounds with m/z values of 729, 757, and 785 that were apparently related to anionogenic lipids of the diacylglyceryl family were found in all of the samples. Common phospholipids and ceramides (Cers) were not present among the major MGS lipids. Phosphocholine-based lipids were found in MGS in quantities less than 0.01% (w/w), if at all. This ratio is two orders of magnitude lower than reported previously. These observations suggest that MGS are a major source of nonpolar lipids of the WE and Chl-E families for the tear film lipid layer, but not of its previously reported (phospho)lipid, Cer, and fatty acid amide components.